Nascent Synthesis of Leader Sequence-Containing Subgenomic Mrnas in Coronavirus Genome-Length Replicative Intermediate RNA

Virology 275, 238–243 (2000) doi:10.1006/viro.2000.0489, available online at http://www.idealibrary.com on View metadata, citation and similar papers at core.ac.uk brought to you by CORE

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Nascent Synthesis of Leader Sequence-Containing Subgenomic mRNAs in Coronavirus Genome-Length Replicative Intermediate RNA

Tetsuya Mizutani, John F. Repass, and Shinji Makino1

Department of Microbiology and Immunology, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1019 Received May 12, 2000; returned to author for revision June 6, 2000; accepted June 26, 2000

Infection with coronavirus results in the accumulation of genomic-sized mRNA and six to eight subgenomic mRNAs that make up a 3Ј coterminal nested-set structure. Genome-length negative-strand RNA and subgenomic-length negative-strand RNAs, each of which corresponds to each of the subgenomic mRNAs, also accumulate in infected cells. The present study examined whether the genome-length negative-strand RNA serves as a template for subgenomic mRNA synthesis. Genome- length replicative intermediate (RI) RNA was purified by two-dimensional gel electrophoresis of intracellular RNAs from cells infected with mouse hepatitis virus. RNase A treatment of the purified genome-length RI resulted in the production of the genome-length replicative form RNA, indicating that the genome-length RI included genome-length template RNA. RNase protection assays using the purified genome-length RI and two probes, which corresponded to the 5Ј 300-nt region of mRNA 6 and to the same region of mRNA 7, showed the presence of nascent leader sequence-containing subgenomic mRNAs in the genome-length RI. These data demonstrated that the genome-length negative-strand RNA serves as a template for subgenomic mRNA synthesis. © 2000 Academic Press

Coronavirus is an enveloped RNA virus containing a virus-infected cells. Coronaviruses and arteriviruses are large positive-strand genomic RNA. Coronavirus-infected classified under the newly established order Nidovirales; cells produce a genomic-sized mRNA, mRNA 1, and six both viruses probably use similar transcription strate- to eight species of successively smaller subgenomic gies. mRNAs, which form a 3Ј coterminal nested-set structure One of the unsolved questions in nidovirus transcrip- (1–3). Characteristics of the unique coronavirus nested- tion is whether the template for subgenomic mRNAs is of set structure are that all the mRNAs are polyadenylated genome length or subgenome length. Two major models at the 3Ј-end; the messages are successively larger by have been proposed. One suggests that a genome- Ј Ј one gene at the 5 -end with only the 5 -most gene on the length negative-strand RNA serves as a template for all polycistron being translated; and each has a 60- to 90- the mRNAs (11), while the other model proposes that Ј nt-long leader sequence at the 5 -end (4–6). The leader each negative-strand subgenomic RNA is a unique tem- Ј sequences are derived from the 5 -end of genomic RNA plate for synthesis of the correspondingly sized sub- (the only place on the genome where they are encoded) genomic mRNA (8, 12–15). A study using mouse hepatitis and, by an unknown type of discontinuous synthesis (7), virus (MHV), a prototypic coronavirus, demonstrated that they join the 5Ј-ends of each of the messages at the early in infection negative-strand genomic RNA, and not intergenic regions. Infected cells also produce negative- negative-strand subgenomic RNA, is the template for strand genomic RNA and subgenomic RNAs, each of subgenomic mRNA synthesis (16); this study did not which corresponds to one mRNA species (8). These determine the template late in infection. negative-strand RNAs contain a poly (U) sequence at the Here we describe whether MHV negative-strand 5Ј-end and an anti-leader sequence at the 3Ј-end (9). genomic RNA is also the template for subgenomic mRNA Like coronaviruses, cells infected with arteriviruses produce a 3Ј coterminal nested set of mRNAs with the synthesis late in infection. Previous studies of replicative leader sequence at the 5Ј-end of the mRNAs (10). Sub- intermediate (RI) RNAs in MHV-infected cells were based genomic negative-strand RNAs that correspond to each on the detection of two different types of RI RNAs: sub- of the subgenomic mRNAs are also produced in arteri- genomic RIs and genome-length RI. After a short pulse of radiolabeling, the subgenomic RIs (13) appear as six to seven discrete RNA bands when separated by using 1 To whom reprint requests should be addressed. Fax: (409) 772- nondenaturing agarose gel electrophoresis. The polarity 5065. E-mail: [email protected]. of these elongating RNAs in the subgenomic RIs has not

FIG. 1. Two-dimensional gel electrophoresis of the poly(A)-minus fraction of MHV-intracellular RNAs. 32P-labled MHV-infected intacellular RNAs that did not bind to oligo-dT cellulose were mixed with lambda HindIII marker DNA fragments and separated on 0.8% nondenaturing two-dimensional gel electrophoresis. (A) Ethidium bromide staining of the gel after two-dimensional gel electrophoresis. Lambda HindIII marker DNA fragments and ribosomal RNAs (28S rRNA and 18S rRNA) are shown. (B) Autoradiogram of the two-dimensional gel. The box region containing genome-length RI was excised from the gel and used for further study. The asterisk is an artifact of the gel electrophoresis. A low level of mRNA 7, which was not completely removed by oligo-dT cellulose column chromatography, is also shown. been identified. The genome-length RI RNA appears as a MHV subgenomic mRNAs. A strong diffuse radioactive slowly migrating diffuse band on a nondenaturing elec- RNA signal, which migrated slower than the 24-kbp-long trophoretic gel (11, 13). RNase A treatment of the ge- lambda HindIII marker DNA fragment, was detected after nome-length RI RNA results in the production of replica- two-dimensional gel electrophoresis. The gel region that tive-form (RF) RNA 1, which comigrates with mRNA 1 (11, migrated more slowly than the 24-kbp-long DNA frag- 13), demonstrating that the nascent RNAs are elongating ment was excised carefully (Fig. 1). Based on a previous on genome-length template RNA in the genome-length study of the genome-length RI (13), this region of the gel RI. RNase T1 fingerprinting analysis showed that the should contain the genome-length RI. majority of elongating RNAs in the genome-length RI are We further separated the RNA in the gel slices under positive-sense (11), strongly indicating that the genome- different electrophoretic conditions to estimate the purity length RI RNA must contain a genome-length negative- and the properties of the gel-purified genome-length RI. strand RNA template. Accordingly, if the negative-strand First we looked for contamination of subgenomic mRNAs genome-length MHV RNA is a template for subgenomic in the gel-purified genome-length RI by processing the mRNA synthesis, then all the subgenomic mRNAs are gel piece obtained after two-dimensional gel electro- likely to be elongating in a genome-length RI RNA. phoresis in a new, nondenaturing 0.8% TBE gel next to To test for the possibility that subgenomic mRNAs are 32P-labled MHV intracellular RNAs (Fig. 2A). Electro- elongating from the negative-strand genome-length MHV phoresis of the purified gel piece resulted in a diffuse RNA in the genome-length RI RNA, we first purified slowly migrating signal whose migration pattern was genome-length RI RNA. DBT cells were infected with the similar to the published MHV genome-length RI RNA (11, A59 strain of MHV at a multiplicity of infection of 20; 13). The purified gel pieces did not contain MHV mRNAs defective interfering RNAs were undetectable in this even after a long exposure of the gel (data not shown). stock of MHV (13). The MHV RNAs were radiolabeled Next we examined the production of the RF1 RNA after from 4 to 7 h postinfection (p.i.) with 32Pi in the presence RNase A treatment of the purified gel pieces. The gel of actinomycin D as described previously (17). Intracel- piece was treated with RNase A (0.33 ␮g/ml) in a buffer lular RNA was extracted at 7 h p.i. using the Totally RNA (233 mM NaCl, 3.3 mM Tris–HCl, pH 7.4, 10 mM EDTA) for kit (Ambion), treated with DNase I, and applied onto an 5 min at room temperature. After RNase A treatment, the oligo-dT cellulose chromatography column. The RNA gel piece was soaked in TBE buffer for 10 min at room fraction that did not bind to the oligo-dT cellulose was temperature. Then the gel piece was placed into the well collected; this step lowered the amount of poly (A)-con- of a 0.8% TBE agarose gel and electrophoresed along- taining mature MHV mRNAs in the RNA preparation. side labeled MHV intracellular RNAs (Fig. 2B), which Then the RNA sample was applied to a two-dimensional resulted in a single band that roughly comigrated with 0.8% TBE (89 mM Tris, 89 mM boric acid, 2 mM EDTA) mRNA 1. Subgenomic RF RNAs were not detected even agarose gel containing ethidium bromide (Fig. 1). We after much longer exposure of the gel. The migration of hoped that the two-dimensional gel electrophoresis this RNase-A-resistant band was very similar to that of would efficiently separate genome-length RI RNA from the RF 1 RNA described previously (11, 13). We further 240 RAPID COMMUNICATION

two nonradiolabeled negative-sense RNA probes, one complementary to the 5Ј 300-nt region of mRNA 6 (probe 1) and the other to the same region in mRNA 7 (probe 2), were independently mixed with heat-denatured 32P-labeled purified genome-length RI. After hybridization and subsequent RNase A and RNase T1 treatment, RNase- resistant RNA fragments were detected on electrophoretic sequence gels. If leader-sequence-containing subgenomic mRNA 6 elongates on the genome-length RI, then the RNase protection assay using probe 1 and purified radiolabeled genome-length RI should produce a radiolabeled 300-nt-long RNA fragment (300-nt fragment) that corresponds to the 5Ј-end 300 nt of nascent mRNA 6 (see Fig. 3A). In addition, two RNA bands of approximately 70 and 240 nt long (240-nt fragment) should be detected, the former corresponding to the leader sequence of all the nascent mRNAs other than mRNA 6 and the latter to the 5Ј 240-nt region of the mRNA 6 body sequence, which is found in nascent MHV mRNAs from 1 through 5. Similar results were expected FIG. 2. Characterization of genome-length RI. (A) Nondenaturing for the RNase protection assay using probe 2; in this agarose gel electrophoresis of purified gel pieces isolated after two- case a 300-nt-long radiolabeled RNA fragment should dimensional agarose gel electrophoresis (lane 2). Total intracellular represent nascent leader-sequence-containing mRNA 7 RNAs from MHV-infected cells (lane 1). (B) Nondenaturing agarose gel electrophoresis of purified gel pieces after RNase A treatment (lane 2). (Fig. 3A). If only genome-length mRNA 1 elongates on Total intracellular RNAs from MHV-infected cells (lane 1). (C) Agarose genome-length RI, then the RNase protection assays gel electrophoresis of an RNA sample that was extracted from the using either probe should yield just a 70-nt-long band purified gel pieces under the denaturing condition (lane 2). Total intra- and a 240-nt-long band. cellular RNAs from MHV-infected cells (lane 1). Two PCR products, each corresponding to the most 5Ј 300 nt of mRNA 6 and mRNA 7, were cloned into a TA confirmed the presence of the genome-length RI RNA in cloning vector (Invitrogen). Probe 1 and probe 2, the the purified gel piece under denaturing gel electrophore- nonradiolabeled negative-strand RNA transcripts, were sis. RNAs were extracted from the gel-purified gel pieces transcribed from cloned plasmids using the T7 mMES- using the RNaid kit (BIO 101) and then separated using SAGE mMACHINE kit (Ambion). In a separate experi- denaturing formaldehyde gel electrophoresis (Fig. 2C). ment, 32P-labeled genome-length RI was prepared using We only detected a smeary signal, the size of which two-dimensional gel electrophoresis as described ranged from genomic in length to less than the size of above. RNAs were extracted from the gel pieces by using mRNA 7; we saw no discrete MHV mRNAs. Incubation of the RNaid kit, heat denatured at 65°C for 10 min, and genome-length RI at 65°C for 10 min and subsequent quickly chilled on ice. Labeled RNAs were then applied separation in a 0.8% TBE agarose gel also produced a to an oligo-dT cellulose chromatography column. We similar smeary RNA signal (data not shown). We inter- collected the RNA fraction that bound to the oligo-dT preted this absence of discrete mRNAs with the pres- cellulose, the poly(A)-containing fraction, and the fraction ence of a smeary signal to mean that the smear repre- that did not bind to the oligo-dT cellulose, the poly(A)- sented nascent RNAs that were elongating in genome- minus fraction. We assumed that the poly(A)-containing length RI. Other experiments showed poor recovery of fraction included nascent poly(A)-containing mRNAs RNA molecules larger than mRNA 3 from a gel, while elongating in the genome-length RI and mature mRNAs recovery of small RNAs was excellent (unpublished that might be contaminating the purified genome-length data). This reduced efficiency of large RNA recovery from RI preparation; and we assumed that the poly(A)-minus the first gel appears as a drop in signal above the size of would include mostly nascent RNA molecules and pos- mRNA 3 in the second separation (Fig. 1C). We repeated sibly be contaminated with degraded mature MHV RNAs these experiments at least three times and obtained that lacked the poly(A) sequence. We separately mixed consistent results. We purified genome-length RI without each of the RNA fractions with each of the probes and contamination of any detectable level of MHV sub- performed the RNase protection using the RiboQuant kit genomic mRNAs. (Pharmingen), according to the manufacturer’s instruc- We used RNase protection assays to look for nascent tion. After RNase A and T1 treatment, the samples were subgenomic mRNAs containing the leader sequence in incubated with proteinase K and extracted using phenol– the genome-length RI. For these assays, an excess of chloroform, and the RNAs were precipitated with etha- RAPID COMMUNICATION 241

FIG. 3. RNase protection assay. (A) Schematic diagram of RNase protection assay. Binding sites of probe 1 and probe 2 to MHV mRNAs are shown. Shaded boxes represent the leader sequence. (B) RNase protection assay using total intracellular RNA from MHV-infected cells (lane 1), poly(A)-containing fraction of the purified genome-length RI (lane 2), and poly(A)-minus fraction of the purified genome-length RI (lane 3). RNA size marker (lane M). Probe 2 was used for RNase protection assay. The 300-nt fragment and the 240-nt fragment are indicated by an arrow and an arrowhead, respectively. (C) RNase protection assay using total intracellular RNA from MHV-infected cells (lane 1), poly(A)-containing fraction of the purified genome-length RI (lane 2), and poly(A)-minus fraction of the purified genome-length RI (lane 3). Probe 1 was used for RNase protection assay. The 300-nt fragment and the 240-nt fragment are indicated by an arrow and an arrowhead, respectively. nol. Finally the RNAs were applied to a 6% polyacrylami- tently detected 300- and 240-nt fragments using the de–urea gel. Control assays combining 32P-labeled intra- poly(A)-minus fraction, while these bands were underde- cellular RNAs from MHV-infected cells with probe 2 and tectable or detected at a very low level using the poly(A)- with probe 1 both yielded the 300- and the 240-nt frag- containing fraction. ments (Figs. 3B and 3C). Production of these two ex- If the purified genome-length RI were contaminated pected fragments demonstrated that this RNase protec- with a large amount of mRNA 6 and mRNA 7, then we tion assay could specifically distinguish mRNA 6 and should have detected the 300-nt-long fragment in the mRNA 7 from intracellular viral mRNA species. The poly(A)-containing fraction. The underdetectable levels of 240-nt fragment appeared as two closely migrating dis- the 300-nt-long fragment and the 240-nt fragment in the crete bands (Fig. 3B); a shorter exposure of Fig. 3C, lane poly(A)-containing fraction indicated that contamination 1, also showed two closely migrating discrete bands with mRNAs was very low in the gel-purified genome- making up the 240-nt fragment. The presence of se- length RI. Consistent with RNase protection assay data, quence heterogeneity at the leader–body fusion site of contamination of mature MHV subgenomic mRNAs in MHVmRNA6(18) may be the reason for the two closely purified genome-length RI was at an underdetectable migrating 240-nt fragments in the RNase protection as- level when purified genome-length RI was separated by say using probe 2. However, the origin of two 240-nt agarose gel electrophoresis (Fig. 2). In contrast, we fragments in the RNase protection assay using probe 1 clearly detected the 300-nt fragment in the poly(A)-minus is unclear, because extensive sequence heterogeneity fraction with either probe 1 or probe 2. As discussed does not exist at the leader–body fusion site of mRNA 7 above, this 300-nt fragment could be produced from (18). It is possible that the RNase protection assay did nascent leader-sequence-containing mRNA 6 and mRNA not quantitatively reflect the actual amounts of individual 7 and/or from degraded MHV RNAs lacking the poly(A) intracellular viral mRNA species. The RNase protection sequence that might be residually present in the purified assay using the poly(A)-minus fraction of the purified genome-length RI. Our preparation of MHV intracellular genome-length RI and probe 1 as well as the same RNA RNA showed minimal RNA degradation (Fig. 2A); there- fraction and probe 2 both showed the 300-nt fragment fore, in our RNase protection assays we think that the and the 240-nt fragment (Figs. 3B and 3C). In contrast, possibility that the 300-nt fragment was solely produced neither the 300-nt nor the 240-nt fragment was detected from contaminating degraded mRNAs is highly unlikely. when the poly(A)-containing fraction of the purified ge- More likely, the 300-nt fragment in the poly(A)-minus nome-length RI was used in combination with probe 2 or fraction was produced from nascent subgenomic mRNA with probe 1. In three repeated experiments, we consis- 6 and mRNA 7, both of which contain the leader se- 242 RAPID COMMUNICATION quence. Theoretically, nascent leader-sequence-contain- genomic RNA is a template for subgenomic mRNA syn- ing mRNAs 6 and 7, which are shorter than 300 nt in thesis throughout the infection. length, should produce a smeared RNA signal in the RNase A treatment of MHV genome-length RI, in which RNase protection assay. We could not detect the ex- nascent subgenomic mRNAs are elongating on a ge- pected smeared RNA signals, so we supposed that the nome-length negative-strand RNA, resulted in RF 1. In signal was too low to be detected under the present contrast, alphaviruses, which produce a subgenomic experimental conditions; in both autoradiograms, gels mRNA that corresponds to the 3Ј-region of viral genome, were exposed to X-ray films for 3 weeks, and further produce three RF RNAs after RNase A treatment of al- exposure had little effect on signal detection. RNase A phavirus RI RNA; with this virus, genome RNA and sub- treatment of the gel-purified genome-length RI produced genomic RNA elongate on a genome-length negative- RF 1 (Fig. 2B), and the RNase protection assay demon- strand RNA in its RI RNA (19). Why RNase A treatment of strated the presence of nascent leader-sequence-con- coronavirus genome-length RI produces RF 1 but not taining subgenomic mRNAs in the genome-length RI. subgenome-length RFs is unknown. Apparently, leader-sequence-containing subgenomic Although the present study and the previous study (16) mRNAs were produced from the genome-length nega- established that negative-strand genomic RNA is a tem- tive-strand RNA in the genome-length RI. These data are plate for MHV subgenomic mRNA synthesis throughout consistent with the model of coronavirus discontinuous the infection, these studies do not eliminate the possi- transcription (7, 11), which involves fusion of a leader bility that MHV subgenomic mRNA is also synthesized sequence to every mRNA body during subgenomic from subgenomic negative-strand template RNA late in mRNA synthesis. infection. A number of studies aimed to test whether The characteristics of the genome-length RI that we positive-strand RNA synthesis occurs using subgenomic described here agree with earlier analyses of MHV ge- negative-strand template RNA in cells infected with coro- nome-length RI (11, 13). Sawicki and Sawicki (13) de- naviruses (8, 12–14) and with arterivirus (15), yet did not scribed a slowly migrating genome-length RI during elec- provide direct evidence of positive-strand RNA elonga- trophoresis of intracellular RNA from MHV-infected cells tion from the subgenomic-length negative-strand tem- in a nondenaturing gel. Their treatment of the gel fraction plate. Sawicki and Sawicki revealed the presence of containing the genome-length RI with RNaseA gave subgenomic RIs in MHV-infected cells (13), and their data mostly RF 1 and a low level of subgenome-length RF directly demonstrated elongation of nascent RNA on RNAs, RF 2 and RF 3; RF 2 and RF 3 may well have been subgenomic-length template in infected cells, however, due to the presence of contaminating subgenomic RIs in the polarity of the template RNA in the subgenomic RIs the genome-length RI preparation. Baric et al. also was not described. It is interesting to note that sub- showed that genome-length RI RNA purified by Sepha- genomic RIs appear as discrete RNA bands that comi- rose 2B-CL column chromatography migrates slowly on grate with subgenomic RFs, which were produced after nondenaturing gels appearing as a smeared RNA and RNase A treatment of subgenomic RIs (13); electro- produces RF 1 after RNase A treatment (11). The authors phoretic patterns of subgenomic RIs imply that sub- compared RNase T1 fingerprinting patterns of column- genomic RI exists as a completely double-stranded form. purified genome-length RIs under two conditions. Under Because RI RNAs that are efficiently synthesizing nas- one condition the genome-length RI was digested with cent RNA molecules appear as a diffuse RNA signal in RNase T1 without denaturation and under the other con- nondenaturing gels, multiple nascent RNAs may not be dition the genome-length RI was digested after denatur- produced in MHV subgenomic RIs. Instead, subgenomic ation of RNA. They found that T1 spots corresponding to RIs may exist as complete double-stranded RNAs, in leader sequence and the leader–body fusion site for which newly synthesized subgenome-length RNA of ei- subgenomic mRNAs appear as strong spots in the sam- ther polarity hybridizes with its subgenomic-length tem- ple treated under the nondenaturing condition (11). plate RNA. Recently, Baric and Yount reported the kinetic These data led the authors to propose that leader-se- radiolabeling analysis of MHV intracellular RNAs (12). quence-containing subgenomic mRNAs elongate on ge- They demonstrated that the incorporation of radioiso- nome-length RI. Although the previous study using the topes into a subgenomic RF RNA, RF 7, increases rapidly column-purified genome-length RI (11) and the present during 5-min pulse-labeling of MHV-infected cells; MHV study used different experimental approaches to exam- RF RNAs were obtained after RNase treatment of 32P- ine the nascent RNAs in the genome-length RI, the data labeled MHV intracellular RNAs. The authors interpreted from these two independent studies indicated that MHV these data that the incorporation of radioisotopes into genome-length negative-strand RNA is the template RNA nascent positive-strand RNAs, which are elongating on for subgenomic mRNA synthesis. We showed that neg- subgenomic negative-strand template RNA in the sub- ative-strand genomic RNA is the template for subgenomic RI, is saturated quickly. They also showed the genomic mRNA synthesis early in infection (16). Our new gradual increase of the RF 7 radioactivity after 5-min data strongly support the idea that negative-strand pulse-labeling, which they interpreted to represent the RAPID COMMUNICATION 243 synthesis of a constant and low level of subgenomic 5. Lai, M. M., Baric, R. S., Brayton, P. R., and Stohlman, S. A. (1984). negative-strand RNA, which is used as a template for Characterization of leader RNA sequences on the virion and subgenomic mRNA synthesis. These interpretations of mRNAs of mouse hepatitis virus, a cytoplasmic RNA virus. Proc. Natl. Acad. Sci. USA 81(12), 3626–3630. the data led the authors to claim that nascent positive- 6. Spaan, W., Delius, H., Skinner, M., Armstrong, J., Rottier, P., Smeek- strand RNAs are elongating on subgenomic negative- ens, S., van der Zeijst, B. A., and Siddell, S. G. (1983). Corona- strand template RNA in the subegenomic RI (12). How- virus mRNA synthesis involves fusion of non-contiguous se- ever, their data can be interpreted differently such that quences. EMBO J. 2(10), 1839–1844. subgenomic RIs are continuously produced efficiently in 7. Jeong, Y. S., and Makino, S. (1994). Evidence for coronavirus dis- infected cells, whereas many of them are quickly de- continuous transcription. J. Virol. 68(4), 2615–2623. 8. Sethna, P. B., Hung, S. L., and Brian, D. A. (1989). Coronavirus graded. Because subgenomic RIs may exist as complete subgenomic minus-strand RNAs and the potential for mRNA double-stranded RNAs, subgenomic RIs can be an ex- replicons. Proc. Natl. Acad. Sci. USA 86(14), 5626–5630. cellent interferon inducer; efficient accumulation of sub- 9. Sethna, P. B., Hofmann, M. A., and Brian, D. A. (1991). Minus-strand genomic RIs in infected cells may elicit the interferon copies of replicating coronavirus mRNAs contain antileaders. activation pathway. It is not surprising that MHV has J. Virol. 65(1), 320–325. developed a mechanism that degrades most sub- 10. Snijder, E. J., and Meulenberg, J. J. (1998). The molecular biology of genomic RIs to inhibit double-strand RNA accumulation arteriviruses. J. Gen. Virol. 79(Pt. 5), 961–979. 11. Baric, R. S., Stohlman, S. A., and Lai, M. M. (1983). Characterization in infected cells. None of the published studies demon- of replicative intermediate RNA of mouse hepatitis virus: Pres- strated the polarity of nascent RNAs elongating on sub- ence of leader RNA sequences on nascent chains. J. Virol. 48(3), genomic RIs in nidovirus-infected cells, hence it is pos- 633–640. sible that nascent negative-strand RNAs are elongating 12. Baric, R. S., and Yount, B. (2000). Subgenomic negative-strand RNA on subgenomic RIs. The data presented by Baric and function during mouse hepatitis virus infection. J. Virol. 74(9), Yount (12) do not exclude the possibility that subgenomic 4039–4046. 13. Sawicki, S. G., and Sawicki, D. L. (1990). 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